Evaluation of Drug Plasma Stability Using an Ultra-High Throughput Laser Diode Thermal Desorption (Ldtd) Methodology Introduction The Laser Diode Thermal Desorption (LDTD) interface with tandem mass spectrometry is a new technology allowing an ultra-high throughput sample analysis (approximately 10 seconds per sample). Unlike Matrix-assisted Laser Desorption/Ionization (MALDI), LDTD does not require enhancing matrix to assist compound ionization. The LDTD/MS/MS analysis does not utilize organic mobile phase and liquid chromatography. We performed plasma stability experiments to examine the applicability of this technology using twelve compounds with different chemical properties. Collected plasma stability samples were analyzed by LDTD/MS/MS and LC/MS/MS which was considered as a gold standard approach. The chemical structures and the halflives of these compounds were evaluated by these two methodologies for a comparison.
Chemicals
Experimental ▲▲
A group of 12 compounds were incubated with human and rat plasma at 5 μM for 0, 10, 30, 60 and 120 minutes at 37 °C, respectively.
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At each time point, 100 µL of 5 µM plasma sample was taken and was added to 400 µL of 1 µM carbutamide/ACN solution in a 96-deep well plate.
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After protein precipitation, supernatants were evenly split for analysis.
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For LC/MS/MS analysis, supernatants were diluted with the mobile phase A (95/5 H2O/ACN, 0.1% formic acid) prior to analysis.
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For LDTD/MS/MS analysis, 2 µL of supernatants were transferred into a 96Lazwell plate. The solvent was allowed to evaporate at room temperature (less than 2 minutes) before the analysis.
MRM Transitions
Human Plasma Stability Data (% Remaining)
Ming-Chih D. Ho , Ming-Xiang Liao ; 2 1 Cindy Xia , and Lily Li 1
2
Tandem Labs New England Millennium Pharmaceuticals2 1
Chemical Properties of Compounds Used for LDTD/MS/MS Analysis
LC/MS/MS ▲▲
Column: Phenomenex Hydro-RP C18 (50 x 2.0 mm)
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Column Heater: 40 g °C
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Mobile Phase A: 95/5 H2O/ACN, 0.1% formic acid Mobile Phase B: 10/90 H2O/ACN, 0.1% formic acid
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Injection Volume: 10 µL
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Gradient
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API 5000 (Applied Biosystem/Sciex)
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MRM under an electrospray positive mode
LDTD/MS/MS ▲▲
A Phytronix LDTD source (model T-960) on Thermo TSQ Vantage triple quadrupole mass spectrometer
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Two micro-liters of supernatants were deposited onto the LazWell plate.
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Dry the solvent on the LazWell plate prior to LDTD/MS/MS analysis
Results and Discussion Results and Discussion
Representative LDTD/MS/MS Thermal Desorption Peaks
Rat Plasma Stability Data (% Remaining)
Chemistry vs. Ionization: We found that the ionization of basic compounds, such as amine containing compounds are generally very good by using the LDTD/ MS/MS method. The LDTD source produced strong signals for the hetero-cyclic compounds (e.g., Albendazole). Ionization of Albendazole was weak using an electrospray source on API 4000. However, for quaternary amines, the LC/MS/MS method has an advantage over the LDTD/MS/MS method. The presence of OH groups seemed to weaken the LDTD ionization. A signal could not be obtained when using the LDTD/MS/MS for compounds possessing a carboxylic acid functional group, e.g., Cerivastatin (data not shown) and Methotrexate. Throughput: The total analysis time for 100 samples was less than 20 minutes by LDTD/MS/MS (10 seconds per sample) . It took approximately 5 hours using a 2.5minute LC/MS/MS method. Plasma Stability: The plasma stability data generated from each method matched remarkably well. With each method, we were able to distinguish compounds with a long half-life (> 4hr) from those with a moderate half-life (within 2 hr) and a short half-life (< 10 min) in both human and rat plasma. We found that the stabilities of procaine, eucatropine, diltiazem, propantheline bromide and vinpocetin in rat plasma and in human plasma were different. The findings from both analytical methods also agreed with each other regarding the difference of half-lives of these compounds in these two species.
Conclusion The LDTD/MS/MS technology can be applied as an ultra-high throughput screening method for the stability assessment of small molecules in plasma. The results over 12 compounds suggest a combination of the LDTD/MS/MS method (covering 90% of all screened compound) with the traditional LC/MS/MS method (covering the 10% uncovered by the LDTD/MS/MS) allows to increase the throughput on plasma stability screening of a factor of 6.2 times. The method development of the LDTD/MS/MS analysis of compounds with a carboxylic acid functional group is in progress using different solvents and/or the negative mode.
